Exercise is conceivably the most potent physiological activator of the fuel-sensing enzyme, AMP-activated protein kinase (AMPK), in many tissues in mammals. Extracellular vesicles (EVs) are significant carriers of biologically active cargo that traffic to local or distant organs or cells. We previously demonstrated that EVs containing proteins are released into the bloodstream during exercise and relocate to the liver (1), where they can modify biological processes. In order to define potential mechanism for these actions, we collected blood from both mice and humans, following exercise and isolated EVs using ultracentrifugation (UC) and size exclusion chromatography (SEC). The presence of EVs was confirmed using nanoparticle tracking analysis, in both SEC-derived mouse EVs (A) and UC-derived human EVs (B). Phospho-AMPK and total-AMPK were detected in SEC-derived mouse EV-rich fractions in both sedentary and exercise EVs and absent in the EV poor-fractions (C). We examined AMP-activated kinase activity in both sedentary and exercise samples from mice and humans. In both species, exercise-derived EV-rich fractions following sonication, induced a large increase in kinase activity compared with sedentary EV-rich fractions (D-E). Importantly, conducting the activity assay in non-sonicated EV-rich fractions from mice, showed reduced kinase activity implying disruption of the phospholipid membrane on the EV is necessary (E). Exercise-derived EVs irrespective of isolation method or species resulted in increased kinase activity compared with sedentary-derived EVs. We hope to further delineate how AMPK is trafficked into EVs and other potential kinases that may be responsible for this exercise-derived increase in kinase activity.